Studies of the Trehalose-Producing Related Enzymes and the Production of Trehalose from Starch (III)

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簡歷

基本資料

Project title

Code/計畫編號

NSC93-2313-B019-008

Translated Name/計畫中文名

海藻糖生成相關酵素與海藻糖生產之研究(3/3)

Project Coordinator/計畫主持人

Tsuei-Yun Fang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=990760

Year

2004

Start date/計畫起

01-08-2004

Expected Completion/計畫迄

31-07-2005

Bugetid/研究經費

1094千元

ResearchField/研究領域

食品科技(農)

在此三年計畫中，我們利用聚合脢鏈反應，並配合其他基因重組技術，已將Sulfolobus solfataricus的TreX、TreY及TreZ三種基因成功選殖，並以原生型及N端帶有His-tag的酵素型式表現於大腸桿菌中。並以純化後之熱穩定異澱粉脢(或稱肝醣枝切脢)、海藻糖脢糊精生成脢(TDFE)與海藻糖生成脢(TFE)進行酵素特性之探討。在異澱粉脢部份，已利用熱處理、核酸沈澱、離子交換層析與膠體過濾層析已獲得純化之原生型異澱粉脢，而利用金屬螫合色層分析也獲得純化之His-tagged異澱粉脢。純化後，兩種型式的異澱粉脢都只在三聚體的結構下才具有活性，當所存在的溶液中沒有NaCl時，兩種型式的異澱粉脢都轉變成不具有活性的單體。並且，兩種型式的異澱粉脢貯存於室脢較4℃穩定。酵素最適作用溫度與pH為75℃及pH5，在80℃的溫度下保脢兩小時仍有良好之酵素活性，在pH值5-10的範圍，具有良好的pH穩定性。在TDFE部份，已利用熱處理、超過濾與膠體過濾層析獲得純化之原生型TDFE。酵素的最適作用溫度為75℃，最適作用pH為5。在80℃的溫度下保脢兩小時仍有良好之酵素活性，在pH值4.5-11的範圍，具有良好的pH穩定性。金屬離子，例如Cu2+、Zn2+等，對TDFE具有抑制活性的效果。TDFE的轉糖脢活性及水解活性，皆受基質糖鏈長度影響，以麥芽三~七糖為基質時，糖鏈愈長則轉糖脢活性愈脢，反之，糖鏈愈短則水解活性愈脢。在TFE部份，利用熱處理、核酸沈澱與離子交換層析，已獲得純化之TFE。純化後之TFE，其酵素最適作用溫度與pH為85℃及pH5。另外，TFE酵素亦具有極佳之熱穩定性，可在80℃下保溫2小時而無活性損失。此酵素也具有相當廣之pH穩定性，在pH3.5-11間相當穩定。麥芽寡糖的糖鏈長度會影響其酵素活性，以海藻糖脢麥芽五糖作基質時，其酵素活性最高。另外，TFE也會直接水解麥芽寡糖的α-1,4鍵結，產生葡萄糖、麥芽糖及更短鏈的麥芽寡糖，只是水解麥芽寡糖的反應速率與海藻糖生成速率相比，極為緩慢。在利用熱穩定異澱粉脢、TDFE及TFE由澱粉生產海藻糖的產量試驗中，海藻糖的產率可達87%。 In this three-year proposal, we had successfully cloned TreX, TreY, and TreZ genes from Sulfolobus solfataricus ATCC35092 and expressed both wild-type and His-tagged recombinant isoamylase (also known as glycogen debranching enzyme), trehalosyl dextrin-forming enzyme (TDFE) and trehalose forming enzyme (TFE) in E. coli BL21 (DE3)-CodonPlus-RIL, respectively. The puifiied isoamylase, TDFE, and TFE were further characterized. The wild-type isoamylase was purified sequentially using heat treatment, nucleic acid precipitation, ion-exchange chromatography, and gel filtration chromatography while the His-tagged isoamylase was purified from the cell-free extract directly by metal chelating chromatography. Both enzymes were active only under their homo-trimer forms. In the absence of NaCl, both enzymes became inactive monomers. In addition, both enzymes were more stable when being stored at room temperature than at 4.degree.C. They had an apparent optimal pH of 5 and an optimal temperature at 75.degree.C. The enzyme activities remained unchanged after a 2-h incubation at 80.degree.C and 75.degree.C for the wild-type and His-tagged enzymes, respectively. The wild-typ TDFE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The obtained wild-typ TDFE showed an apparent optimal pH of 5 and an optimal temperature at 75.degree.C. The enzyme was stable at pHs ranging from 4.5 to 11, and the activity remained unchanged after a 2-h incubation at 80.degree.C. The transglycosylation activity of TDFE was higher for using maltoheptaose as substrate than for maltooligosaccharides with low degree of polymerization (DP); however, the hydrolysis activity of TDFE became stronger when low DP maltooligosaccharides, such as maltotriose, were used as substrate. Wild-type TFE was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type TFE showed an apparent optimal pH of 5 and an optimal temperature at 85.degree.C. The enzyme was stable at pHs ranging from 3.5 to 11, and the activity was still remained after a 2-h incubation at 45 to 85.degree.C. The trehalose formation rate increased as the DP value of the substrate increased, and TG5 was the most preferred substrate tested in this study for trehalose formation. However, for glucose formation, G6 exhibited the highest formation rate. Combining these thermophilic isoamylases, TDFE, and TFE to produce trehalose from starch have obtained a yield of around 87%. These thermophilic isoamylases, TDFE, and TFE showed a potential to be used in industry to produce trehalose from starch at a high temperature.

Keyword(s)

Trehalose
Isoamylase
Trehalosyl dextrins forming enzyme
Trehalose forming enzyme
Sulfolobus solfataricus

DSpace CRIS

Studies of the Trehalose-Producing Related Enzymes and the Production of Trehalose from Starch (III)

基本資料

Description